C. difficile is a rapidly emerging enteric pathogen which is now the leading cause of nosocomial infectious diarrhea in developed countries. Infections are caused by an anaerobic, spore-forming bacillus that often attacks elderly patients in various healthcare settings following treatment of unrelated infections with antibiotics (Blossom D B, McDonald L C. The challenges posed by reemerging Clostridium difficile infection. Clin Infect Dis. 2007; 45:222-227; Gould C V, McDonald L C. Bench-to-bedside review: Clostridium difficile colitis. Crit. Care. 2008; 12:203). Clinical symptoms range in severity from mild antibiotic-associated diarrhea to a more severe and life-threatening pseudomembranous colitis which if untreated leads to fulminant colitis and death. (Kelly C P, LaMont J T. Clostridium difficile infection. Annu Rev Med. 1998; 49:375-390). Deaths attributable to C. difficile disease have quadrupled in the United States from 5.7 per million persons in 1999 to 23.7 per million in 2004 (Redelings M D, Sorvillo F, Mascola L. Increase in Clostridium difficile-related mortality rates, United States, 1999-2004. Emerg Infect Dis. 2007; 13:1417-1419). Estimates of the cost for treatment for CDI in the United States have been dramatically revised upward from $1 billion in 2002 to $3.2 billion in 2007, due to a dramatic increase in the number of cases and increasing severity of the disease (O'Brien J A, Lahue B J, Caro J J, Davidson D M. The emerging infectious challenge of Clostridium difficile-associated disease in Massachusetts hospitals: clinical and economic consequences. Infect Control Hosp Epidemiol. 2007; 28:1219-1227). Rapidly emerging hypervirulent antibiotic-resistant strains of C. difficile have been associated with recent epidemics of CDI in North America and Europe with increased morbidity and mortality in healthcare settings (Warny M, Pepin J, Fang A et al. Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet. 2005; 366:1079-1084). However, epidemic strains of C. difficile are being increasingly reported in community-acquired disease in which no previous history of antibiotic use was evident. Since epidemic strains of C. difficile spores have now been repeatedly detected in commercial beef, poultry, and other food products, increasing attention is now being focused on food products as one possible source of community infection (Rodriguez-Palacios A, Staempfli Duffield T, Weese J S. Clostridium difficile in retail ground meat, Canada. Emerg Infect Dis. 2007; 13:485-487). Although primary CDI can be successfully treated with metronidazole or vancomycin, recurrent episodes of antibiotic-resistant CDI complicate management, and development of a vaccine against infection with Clostridium difficile could be useful to prevent relapse (Musher D M, Nuila F, Logan N. The long-term outcome of treatment of Clostridium difficile colitis. Clin Infect Dis. 2007; 45:523-524).
One significant challenge in the management of CDI is successful treatment of recurrent disease after resolution of primary disease symptoms. Recurrent CDI (RCDI) typically occurs within 7 days to 3 weeks following cessation of antibiotic treatment for the initial episode. The most significant risk factor for recurrence is recurrence itself (Blossom D B, McDonald L C. The challenges posed by reemerging Clostridium difficile infection. Clin Infect Dis. 2007; 45:222-227). The risk of recurrent infection rises from about 20% after the primary infection to approximately 40% after the first recurrence, further increasing to >60% after two or more recurrences (Kelly C P, LaMont J T. Clostridium difficile—more difficult than ever. N Engl J. Med. 2008; 359:1932-1940). Epidemic strains of C. difficile, including PCR ribotypes 027 and 078, are associated with recurrence and increased severity of disease (Leav B A, Blair B, Leney M et al. Serum anti-toxin B antibody correlates with protection from recurrent Clostridium difficile infection (CDI). Vaccine. 2010; 28:965-969; Goorhuis A, Bakker D, Corver J et al. Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin Infect Dis. 2008; 47:1162-1170; Burns K, Morris-Downes M, Fawley W N, Smyth E, Wilcox M H, Fitzpatrick F. Infection due to C. difficile ribotype 078: first report of cases in the Republic of Ireland. J Hosp Infect. 2010; 75:287-291). In one recent study, these two ribotypes accounted for 54% of typeable isolates from recurrent CDI cases, and 34% of primary cases identified over a one month period (Burns K, Skally M, Solomon K et al. Clostridium difficile Infection in the Republic of Ireland: Results of a 1-Month National Surveillance and Ribotyping Project, March 2009. Infect Control Hosp Epidemiol. 2010). Other risk factors for RCDI include age >65 years, sever underlying illness, and continued use of antibiotics for unrelated disease after resolution of CDI; however, the coincidence of more than one of these factors dramatically increases the probability of recurrence (Kyne L, Wanly M, Qamar A, Kelly C P. Association between antibody response to toxin A and protection against recurrent Clostridium difficile diarrhoea. Lancet. 2001; 357:189-193; Hu M Y, Katchar K, Kyne L et al. Prospective derivation and validation of a clinical prediction rule for recurrent Clostridium difficile infection. Gastroenterology. 2009; 136:1206-1214).
The genome of C. difficile is highly dynamic, and recent studies point to multiple paths leading to the emergence of hypervirulence in epidemic strains (He M, Sebaihia M, Lawley T D et al., Evolutionary dynamics of Clostridium difficile over short and long time scales. Proc Natl Acad Sci USA. 2010; 107:7527-7532). Such genome fluidity suggests that modulation of virulence would be expected, with variation shaped through positive selection by host immunity as well as clinical pressures including routine antibiotic therapy (He M, Sebaihia M, Lawley T D et al. Evolutionary dynamics of Clostridium difficile over short and long time, scales. Proc Natl Acad Sci USA. 2010; 107:7527-7532; Stabler R A, He M, Dawson L et al. Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium. Genome Biol. 2009; 10:R102; Stabler R A, Valiente E, Dawson L F, He M, Parkhill J, Wren B W. In-depth genetic analysis of Clostridium difficile PCR-ribotype 027 strains reveals high genome fluidity including point mutations and inversions. Gut Microbes. 2010; 1:1-8). Genome fluidity seems to explain the repeated isolation of clinical strains of C. difficile in which expected virulence factors are not always present.
Enterotoxins A (TcdA) and B (TcdB) are the primary virulence factors of C. difficile. These toxins are exoenzymes that monoglucosylate small Rho-like GTPases, ultimately leading to the disruption of the actin cytoskeleton of colonic intestinal epithelial cells, destruction of tight junctions, and apoptosis (Voth D E, Ballard J D. Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev. 2005; 18:247-263; Aktories K, Barbieri J T. Bacterial cytotoxins: targeting eukaryotic switches. Nat Rev Microbiol. 2005; 3:397-410). Release of cytokines from intoxicated target cells also leads to massive infiltration of neutrophils into damaged tissue regions, a hallmark of pseudomembranous colitis (Thielman Nathan M., Wilson Kenneth H. Antibiotic-Associated Colitis. In: Mandell G L, Bennett John E, Dolin R, eds. Principles and Practice of Infectious Diseases. 6 ed. Philadelphia: Elsevier Churchill Livingstone; 2005:1249-63). Both enterotoxins are not required to cause disease, and clinical strains in which TcdA is absent have been repeatedly isolated from patients (Kim S J, Kim H, Seo Y et al. Molecular characterization of toxin A-negative, toxin B-positive variant strains of Clostridium difficile isolated in Korea. Diagn Microbiol Infect Dis. 2010; 67:198-201; Pituch H, Brazier J S, Obuch-Woszczatynski P, Wultanska D, Meisel-Mikolajczyk F, Luczak M. Prevalence and association of PCR ribotypes of Clostridium difficile isolated from symptomatic patients from Warsaw with macrolide-lincosamide-streptogramin B (MLSB) type resistance. J Med. Microbiol. 2006; 55:207-213; Pituch H, van LW, Maquelin K et al. Toxin profiles and resistances to macrolides and newer fluoroquinolones as epidemicity determinants of clinical isolates of Clostridium difficile from Warsaw, Poland. J Clin Microbiol. 2007; 45:1607-1610). To date, all clinical isolates of C. difficile express TcdB, and TcdB is the only virulence factor suggested to be specifically required for manifestation of disease in humans (Lyras D, O'Connor J R, Howarth P M et al. Toxin B is essential for virulence of Clostridium difficile. Nature. 2009). Recent studies suggest that TcdB expressed by epidemic strains is hypertoxic due to an extended range of tissue tropism and increased penetration into the cytoplasm of target cells (Lanis J M, Barua S, Ballard J D. Variations in TcdB Activity and the Hypervirulence of Emerging Strains of Clostridium difficile. PLoS Pathog. 2010; 6:e1001061). However, epidemic strains of C. difficile, including PCR ribotypes 027 and 078, typically express both TcdA and TcdB, suggesting that co-expression of both toxins may play an important role in the severity of disease caused by these epidemic strains.
In addition to toxins A and B, PCR-ribotypes 027 and 078 also invariably carry an additional toxin affecting the actin cytoskeleton called C. difficile transferase (Cdt); this toxin has also been called binary toxin because it is composed of a catalytic A subunit and a cell-binding B subunit (Perelle S, Gibed M, Bourlioux P, Corthier G, Popoff M R. Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196. Infect Immun. 1997; 65:1402-1407; Rupnik M, Grabnar M, Geric B. Binary toxin producing Clostridium difficile strains. Anaerobe. 2003; 9:289-294). The activity of Cdt causes rearrangement of the actin cytoskeleton of intestinal epithelial cells, disrupting tight junctions and allowing better penetration and binding of toxin B to basolateral receptors, possibly enhancing the virulence of epidemic strains (Carter G P, Rood J I, Lyras D. The role of toxin A and toxin B in Clostridium difficile-associated disease. Gut Microbes. 2010; 1:58-64). Surprisingly, it was recently discovered that Cdt also appears to enhance colonization of the intestinal tract by inducing microtubule-based protrusions which enhance the adherence of C. difficile (Schwan C, Stecher B, Tzivelekidis T et al. Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria. PLoS Pathog. 2009; 5:e1000626).
Since antibiotic use is a major contributing factor to the occurrence of CDI, a non-antibiotic vaccine-based approach for preventing disease could potentially reduce patient morbidity and mortality due to recurrent infection following cessation of antibiotic treatment. Recent economic computer models strongly indicate that development of a vaccine against infection with C. difficile could be cost-effective over a wide range of vaccine efficacies and costs when used to prevent recurrent disease (Lee B Y, Popovich M J, Tian Y et al. The potential value of Clostridium difficile vaccine: an economic computer simulation model. Vaccine. 2010; 28:5245-5253). However, no such vaccine is currently on the market.
Reduced serum IgG antibody responses to C. difficile toxin A has been proposed as a risk factor linked to recurrence of infection with C. difficile (Kyne L, Wamy M, Qamar A, Kelly C P. Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A. N Engl J. Med. 2000; 342:390-397; Aboudola S, Kotloff K L, Kyne L et al. Clostridium difficile vaccine and serum immunoglobulin G antibody response to toxin A. Infect Immun. 2003; 71:1608-1610), and evidence from a recent Phase 2 clinical trial indicates that lower serum concentrations of neutralizing antibody against both TcdA and TcdB are associated with recurrence of CDI (Leav B A, Blair B, Leney M et al. Serum anti-toxin B antibody correlates with protection from recurrent Clostridium difficile infection (CDI). Vaccine. 2010; 28:965-969). Additional data from a related Phase 2 trial showed that co-administration of antibiotics to patients with CDI, along with humanized IgG monoclonal antibodies neutralizing toxin A (CDA1) and toxin B (CDB1), provided significant protection against recurrent disease, with patients suffering from multiple recurrences being particularly likely to benefit (Lowy I, Molrine D C, Leav B A et al. Treatment with monoclonal antibodies against Clostridium difficile toxins. N Engl J. Med. 2010; 362:197-205). Sanofi-Aventis has conducted a series of Phase 1 clinical trials testing the immunogenicity of a 3 dose toxoid-based bivalent vaccine targeting both TcdA and TcdB from C. difficile; encouraging serum IgG titers against toxins A and B were observed for both healthy subjects (18-55 years of age) and elderly subjects (≥65), with seroconversion rates of 75% against TcdA and TcdB in both age groups after 3 intramuscular 50 μg doses of toxoid (Foglia, G. ACAM-CDIFFTM: An Active Vaccine Against Clostridium difficile Infection (CDI). 2010. Ref Type: Conference Proceeding). The mechanism(s) by which serum antibody responses are effective against infection and disease caused by C. difficile are unclear, although it has been proposed that entry of IgG antitoxin from the blood into mucosal tissues of the large bowel or intestinal lumen may prevent toxin binding (Kelly C P. Immune response to Clostridium difficile infection. Eur J Gastroenterol Hepatol. 1996; 8:1048-1053; Warny M, Vaerman J P, Avesani V, Delmee M. Human antibody response to Clostridium difficile toxin A in relation to clinical course of infection. Infect Immun. 1994; 62:384-389).
There is a need for new treatments against C. difficile infections, in particular, treatments aimed at preventing recurrent infections of C. difficile in patients. The present invention of a multivalent live Salmonella enterica serovar Typhi (Salmonella Typhi; S. Typhi) vector expressing various toxins from Clostridium difficile satisfies this need.